WO2017015671A1 - Compositions permettant de traiter l'amylose - Google Patents

Compositions permettant de traiter l'amylose Download PDF

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WO2017015671A1
WO2017015671A1 PCT/US2016/043927 US2016043927W WO2017015671A1 WO 2017015671 A1 WO2017015671 A1 WO 2017015671A1 US 2016043927 W US2016043927 W US 2016043927W WO 2017015671 A1 WO2017015671 A1 WO 2017015671A1
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una
strand
monomers
compound
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Kiyoshi Tachikawa
Joseph E. Payne
Padmanabh Chivukula
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Arcturus Therapeutics, Inc.
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Priority claimed from US14/807,223 external-priority patent/US9856475B2/en
Application filed by Arcturus Therapeutics, Inc. filed Critical Arcturus Therapeutics, Inc.
Publication of WO2017015671A1 publication Critical patent/WO2017015671A1/fr
Priority to US15/876,127 priority Critical patent/US10421964B2/en

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Definitions

  • This invention relates to the fields of biopharmaceuticals and therapeutics that are operable by gene silencing. More particularly, this invention relates to the structures, compositions and uses of active agents for inhibiting expression of a TTR gene in a subject.
  • the active agents are UNA oligomers that can be used for gene silencing, and among other things, in methods for treating transthyretin-related amyloidosis.
  • TTR transthyretin
  • TRR transthyretin
  • the pathology of ATTR may include many TTR mutations. Symptoms of
  • ATTR often include neuropathy and/or cardiomyopathy.
  • Peripheral neuropathy can begin in the lower extremities, with sensory and motor neuropathy, and can progress to the upper extremities.
  • Autonomic neuropathy can be manifest by gastrointestinal symptoms and orthostatic hypotension.
  • TTR gene Val-30-Met the most common mutation, have normal echocardiograms. However, they may have conduction system irregularities and need a pacemaker.
  • the ATTR V30M variant can cause lower extremity weakness, pain, and impaired sensation, as well as autonomic dysfunction. Vitreous and opaque amyloid deposits can be characteristic of ATTR.
  • ATTR amyloidosis The major treatment for ATTR amyloidosis is liver transplantation, which removes the major source of variant TTR production and replaces it with normal TTR. Liver transplantation slows disease progression and some improvement in autonomic and peripheral neuropathy can occur.
  • This invention relates to the fields of biopharmaceuticals and therapeutics based on allele selective gene silencing. More particularly, this invention relates to methods for treating transthyretin-related amyloidosis with UNA oligomers capable of allele-selective knockdown of transthyretin.
  • This invention provides UNA oligomers for selectively inhibiting V30M
  • TTR expression which can be used in treating amyloidosis.
  • the UNA oligomers can have a first strand and a second strand, each of the strands being 19-29 monomers in length, the monomers being UNA monomers and nucleic acid monomers.
  • Embodiments include pharmaceutical compositions and methods for treating or preventing TTR-related amyloidosis by administering a UNA oligomer to a subject.
  • Embodiments of this invention include the following:
  • a UNA oligomer for selectively inhibiting V30M TTR expression comprising a first strand and a second strand, each of the strands being 19-29 monomers in length, the monomers comprising UNA monomers and nucleic acid monomers, wherein the oligomer has a duplex structure of from 14 to 29 monomers in length.
  • the UNA oligomer above, wherein the second strand has at least one UNA monomer in the duplex region.
  • the UNA oligomer above, wherein the at least one UNA monomer in the second strand is at any one of positions 2-8 from the 5' end.
  • the UNA oligomer above, wherein the at least one UNA monomer in the second strand is at any one of positions 9-18 from the 5' end.
  • the UNA oligomer above, wherein the at least one UNA monomer in the second strand is at position 6, 7, 15, 16 or 17 from the 5' end.
  • the UNA oligomer above comprising at least one nucleic acid monomer that is base-modified, sugar-modified, or linkage modified.
  • a compound comprising a first strand and a second strand, each of the strands being 19-29 monomers in length, the monomers comprising UNA monomers and nucleic acid monomers, wherein the compound has a duplex region of from 14 to 29 contiguous monomers in length, wherein the first strand is a passenger strand for RNA interference and the second strand is a guide strand for RNA interference, and wherein the compound comprises a sequence of bases targeted to inhibit expression of a TTR gene.
  • the compound may contain from one to seven UNA monomers.
  • the compound may contain a UNA monomer at the 1-end (5' end for non-UNA) of the first strand, a UNA monomer at the second position from the 3' end of the first strand, and a UNA monomer at the second position from the 3' end of the second strand.
  • the compound may contain a UNA monomer at any one or more of positions 2 to 8 from the 5' end of the second strand.
  • the compound may have a 3' overhang comprising one or more UNA monomers, natural nucleotides, non-natural nucleotides, modified nucleotides, or chemically-modified nucleotides, or combinations thereof.
  • the compound may have a 3' overhang comprising one or more deoxythymidine nucleotides, 2'-0-methyl nucleotides, inverted abasic monomers, inverted thymidine monomers, L- thymidine monomers, or glyceryl nucleotides.
  • Embodiments of this invention contemplate compounds in which one or more of the nucleic acid monomers may be a non-natural nucleotide, a modified nucleotide, or a chemically-modified nucleotide.
  • a nucleic acid monomer may have a 2'- O-methyl group, a 2'-methoxyethoxy, or a 2'-deoxy-2'-fluoro ribonucleotide.
  • the compound may not contain fluorine.
  • a compound may have one or more of three monomers at each end of each strand being connected by a phosphorothioate, a chiral phosphorothioate, or a phosphorodithioate linkage.
  • This invention further contemplates a lipid nanoparticle-oligomer compound comprising one or more compounds above attached to the lipid nanoparticle, as well as compositions containing one or more compounds above and a pharmaceutically acceptable carrier.
  • the carrier may include lipid nanoparticles or liposomes.
  • a composition of this disclosure upon administering a single intravenous dose to a subject, may reduce TTR protein in the subject by at least 80% after 10 days, or at least 70% after 20 days, or at least 50% after 30 days.
  • Embodiments of this invention further include methods for treating or preventing TTR-related amyloidosis in a subject in need, by administering to the subject an effective amount of a composition above to the subject.
  • the TTR-related amyloidosis can be ATTR.
  • the administering can be intravenous, subcutaneous, pulmonary, intramuscular, intraperitoneal, or dermal.
  • the effective amount can be a dose of from 0.001 to lO.O mg/kg.
  • the composition upon administering a single dose to a subject, may reduce TTR protein in the subject by at least 70% after 20 days, or by at least 50% after 30 days.
  • This invention includes methods for inhibiting expression of a TTR gene in a subject, by administering to the subject a composition above. Further aspects include the use of a composition above for preventing, ameliorating or treating a disease or condition associated with TTR-related amyloidosis in a subject in need, or for use in medical therapy, or for use in the treatment of the human or animal body.
  • This invention further includes compositions above for use in preparing or manufacturing a medicament for preventing, ameliorating or treating a disease or condition associated with TTR-related amyloidosis in a subject in need.
  • FIG. 1 shows the single nucleotide polymorph (SNP) that exists at position 284 in the V30M mutation mRNA, as compared to the wild type (WT) TTR mRNA.
  • SNP single nucleotide polymorph
  • WT wild type
  • Conventional siRNAs that are complementary to the WT mRNA can be tiled around position 284.
  • FIG. 2 Fig. 2 shows that the conventional siRNAs complementary to the
  • WT mRNA have limited activity in silencing the WT TTR gene, as measured by TTR knockdown in HepG2 cells. Positions 5, 9, 14 and 15, indicated by arrows, appear to be more accessible to silencing than other positions.
  • FIG. 3 shows that conventional siRNAs that are complementary to the V30M mRNA can be tiled around position 284.
  • Four conventional siRNA variations namely V30M-P5, V30M-P9, V30M-P14, and V30M-P15, were prepared.
  • FIG. 4 shows the activity of the four conventional siRNA variations V30M-P5, V30M-P9, V30M-P14, and V30M-P15 measured in the
  • V30M-P5 V30M-P9, and V30M-P15 were more effective against V30M than V30V.
  • the conventional siRNA variation V30M-P14 was not more effective against V30M than V30V.
  • FIG. 5 shows IC50 analysis for the four conventional siRNA variations V30M-P5, V30M-P9, V30M-P14, and V30M-P15 measured in the
  • V30M-P15 variant was 5.6 times more effective against V30M than V30V.
  • selectivity of the conventional siRNAs against V30M over V30V was no more than 5.6.
  • FIG. 6 shows the structure of UNA oligomers that were effective in silencing V30M TTR, as measured in the PSICHECK reporter assay.
  • a first UNA monomer was located at the 5' end of the first strand, also called the passenger strand.
  • the second strand also called the guide strand, formed a duplex region of 19 monomers length with the first strand.
  • Each UNA oligomer had a duplex region of 19 monomers, and a two-monomer overhang at each end.
  • a second UNA monomer was located at the 3' end of the first strand, in the 20 th position, which is in an overhang portion.
  • a third UNA monomer was located at the 3' end of the second strand, in the 20 th position, which is in an overhang portion.
  • P15U6, P15U7, P15U14, P15U15, and P15U16 a fourth UNA monomer was located in the second strand at positions 6, 7, 14, 15 and 16, respectively, counting from the 5' end of the second strand.
  • FIG. 7 shows the activity of UNA oligomers P15U6, P15U7,
  • P15U15, and P15U16 measured in the PSICHECK reporter assay against V30V and V30M gene reporter variants, as compared to the conventional siRNA V30M-P15.
  • the UNA oligomers were more effective against V30M than V30V.
  • the activity of the UNA oligomer P15U6 was substantially and advantageously superior to the activity of the conventional siRNA V30M-P15, where each is targeted to V30M.
  • Fig. 7 shows the surprising and unexpected result that the selectivity of the UNA oligomers, P15U6, P15U7, P15U15, and P15U16, against V30M over V30V was substantially greater than for the conventional siRNA V30M-P15.
  • the selectivity of UNA oligomer PI 5U6 against V30M over V30V was 24, meaning that the IC50 of UNA oligomer P15U6 against V30M (37.6 pM) was 24 times lower than the IC50 of UNA oligomer P15U6 against V30V (919.9 pM).
  • This selectivity was advantageously 4-fold superior to the selectivity of 5.6 shown above in Fig. 5 for the conventional siRNA.
  • FIG. 8 shows the structure of UNA oligomers that were effective in silencing V30M TTR, as measured in the PSICHECK reporter assay.
  • a first UNA monomer was located at the 5' end of the first strand, also called the passenger strand.
  • the second strand also called the guide strand, formed a duplex region of 19 monomers length with the first strand.
  • Each UNA oligomer had a duplex region of 19 monomers, and a two-monomer overhang at each end.
  • a second UNA monomer was located at the 3' end of the first strand, in the 20 th position, which is in an overhang portion.
  • a third UNA monomer was located at the 3' end of the second strand, in the 20 position, which is in an overhang portion.
  • P16U6, P16U7, P16U15, P16U16, and P16U17 a fourth UNA monomer was located in the second strand at positions 6, 7, 15, 16 and 17, respectively, counting from the 5' end of the second strand.
  • FIG. 9 shows the activity of UNA oligomers P16U6, P16U7,
  • FIG. 10 shows the selectivity of UNA oligomer P15U6 against V30M over V30V.
  • the IC50 of UNA oligomer P15U6 against V30M 37.6 pM
  • This selectivity was advantageously 4-fold superior to the selectivity of 5.6 shown in Fig. 5 above for the conventional siRNA.
  • Fig. 10 shows the selectivity of UNA oligomer P15U6 against V30M over V30V.
  • the IC50 of UNA oligomer P15U6 against V30M 37.6 pM
  • This selectivity was advantageously 4-fold superior to the selectivity of 5.6 shown in Fig. 5 above for the conventional siRNA.
  • Fig. 10 shows the selectivity of 5.6 shown in
  • FIG. 11 shows knockdown of Transthyretin protein in vivo as a result of treatment with UNA oligomers targeting Transthyretin TTR mRNA, delivered in a liposomal formulation to male Cynomolgus monkeys. Measurements were made following a single intravenous (IV) dose administration. TTR protein concentration in plasma samples was assessed by LC-MS/MS at various time points. Animals were administered test doses on Day 1 at 0.3 mg/kg. Doses were administered as a 15-minute intravenous (IV) infusion, ⁇ 1 minute, using a pump and disposable syringe.
  • IV intravenous
  • This invention provides UNA oligomers for selectively inhibiting V30M
  • the UNA oligomers of this invention can be used as therapeutics for treating amyloidosis.
  • this invention provides UNA oligomers, compositions and methods for treating transthyretin-related amyloidosis.
  • the UNA oligomers can have a first strand and a second strand, each of the strands being 19-29 monomers in length, the monomers being UNA monomers and nucleic acid monomers.
  • Embodiments of this invention include compositions and methods for treating or preventing TTR-related amyloidosis by administering a UNA oligomer to a subject.
  • the UNA oligomers of this invention are capable of allele-specific knockdown of transthyretin.
  • UNA oligomers are provided for treating amyloidosis related to transthyretin (ATTR).
  • the UNA oligomers of this invention can reduce the depositing of amyloid fibril proteins in various organs and tissues, including the peripheral, autonomic, and central nervous systems.
  • this invention provides therapeutics for ATTR and related amyloid-related diseases.
  • aspects of this invention include UNA oligomers that can be used for treating clinical features of ATTR amyloidosis, including neuropathy and/or
  • UNA oligomers of this invention are targeted to one mutation Val-30-Met TTR.
  • This invention can provide a pharmacological therapy that can undo the formation of TTR amyloid.
  • TTR transthyretin
  • composition of this invention can provide an unexpectedly advantageous duration of action.
  • upon activation of the reaction of the reaction of the reaction of the reaction of the reaction of the reaction of the reaction of the reaction of the reaction can provide an unexpectedly advantageous duration of action.
  • the TTR protein in a subject may be reduced by at least 90% after 20 days.
  • the TTR protein in a subject may be reduced by at least 70% for a period of at least 20 days.
  • the TTR protein in a subject may be reduced by at least 50% for a period of at least 30 days.
  • a subject can be a primate, a human, or other mammal.
  • the UNA oligomers of this invention can be used for inhibiting V30M
  • a UNA oligomer of this invention may have a first strand and a second strand, each of the strands being 19-29 monomers in length.
  • the monomers of a UNA oligomer can include UNA monomers and nucleic acid monomers
  • a UNA oligomer can be a duplex structure of from 14 to 29 monomers in length.
  • the second strand of a UNA oligomer can have at least one UNA monomer in the duplex region.
  • a UNA oligomer can have at least one UNA monomer in the second strand at any of positions 6, 7, 15, 16 or 17 from the 5' end in the duplex region.
  • a UNA oligomer of this invention may have any number of UNA monomers within its total length.
  • a UNA oligomer can include a nucleic acid monomer that is base- modified, sugar-modified, or linkage modified.
  • Embodiments of this invention further provide UNA oligomers that selectively inhibit V30M TTR expression.
  • a UNA oligomer has an IC50 for reducing V30M
  • a UNA oligomer can have a selectivity ratio in vitro of at least 5.
  • the selectivity ratio is the ratio of the IC50 for reducing V30M TTR expression to the IC50 for reducing wild type TTR expression.
  • the selectivity ratio of a UNA oligomer of this invention can range from 2 to 1000. In certain embodiments, the selectivity ratio of a UNA oligomer is at least 2, or at least 5, or at least 10, or at least 30, or at least 30, or at least 50, or at least 100.
  • a UNA oligomer of this invention can selectively inhibit
  • V30M TTR expression in vivo V30M TTR expression in vivo.
  • a UNA oligomer of this invention can selectively inhibit
  • a UNA oligomer can be an active pharmaceutical molecule being a chain composed of monomers, also called an oligomer.
  • the monomers of the oligomer can include UNA monomers and other nucleic acid monomers.
  • the UNA monomers are novel, synthetic molecules that can be attached in a chain to form an oligomer.
  • the nucleic acid monomers can be naturally-occurring nucleotides, modified naturally-occurring nucleotides, or certain non-naturally- occurring nucleotides.
  • a UNA oligomer of this invention is a synthetic, pharmacologically active molecule and can be used in the treatment of a condition or disease.
  • a UNA oligomer of this disclosure can be a double stranded oligomer. Each strand of the double stranded oligomer can be composed of UNA monomers along with a number of nucleic acid monomers for a total length of 19 to 29 monomers.
  • a UNA oligomer of this invention can contain one or more UNA monomers in any strand.
  • the UNA monomers can be in a single strand, or in either strand of a double stranded UNA oligomer, or in both strands of a double stranded UNA oligomer.
  • linker group monomers can be unlocked nucleomonomers (UNA monomers), which are small organic molecules based on a propane-l,2,3-tri-yl-trisoxy structure as shown below:
  • R 1 and R 2 are H, and R 1 and R 2 can be phosphodiester linkages
  • Base can be a nucleobase
  • R 3 is a functional group described below.
  • UNA monomer main atoms can be drawn in IUPAC notation as follows: UNA monomer unit
  • nucleobase examples include uracil, thymine, cytosine, 5- methylcytosine, adenine, guanine, inosine, and natural and non-natural nucleobase analogues.
  • the UNA monomers are not nucleotides, they can exhibit at least four forms in an oligomer.
  • a UNA monomer can be an internal monomer in an oligomer, where the UNA monomer is flanked by other monomers on both sides.
  • the UNA monomer can participate in base pairing when the oligomer is a duplex, for example, and there are other monomers with nucleobases in the duplex.
  • a UNA monomer can be a monomer in an overhang of an oligomer duplex, where the UNA monomer is flanked by other monomers on both sides. In this form, the UNA monomer does not participate in base pairing. Because the UNA monomers are flexible organic structures, unlike nucleotides, the overhang containing a UNA monomer will be a flexible terminator for the oligomer.
  • a UNA monomer can be a terminal monomer in an overhang of an oligomer, where the UNA monomer is attached to only one monomer at either the propane-l-yl position or the propane-3-yl position. In this form, the UNA monomer does not participate in base pairing. Because the UNA monomers are flexible organic structures, unlike nucleotides, the overhang containing a UNA monomer can be a flexible terminator for the oligomer.
  • a UNA monomer can be a flexible molecule
  • a UNA monomer as a terminal monomer can assume widely differing conformations.
  • An example of an energy minimized UNA monomer conformation as a terminal monomer attached at the propane-3-yl position is shown below.
  • UNA oligomers having a terminal UNA monomer are significantly different in structure from conventional nucleic acid agents, such as siRNAs.
  • siRNAs may require that terminal monomers or overhangs in a duplex be stabilized.
  • the conformability of a terminal UNA monomer can provide UNA oligomers with different properties.
  • a UNA oligomer can be a chain composed of UNA monomers, as well as various nucleotides that may be based on naturally- occurring nucleosides.
  • the functional group R 3 of a UNA monomer can be any suitable functional group R 3 of a UNA monomer.
  • R 4 is the same or different for each occurrence, and can be H, alkyl, a cholesterol, a lipid molecule, a polyamine, an amino acid, or a polypeptide.
  • the UNA monomers are organic molecules. UNA monomers are not nucleic acid monomers or nucleotides, nor are they naturally-occurring nucleosides or modified naturally-occurring nucleosides. [0091] A UNA oligomer of this invention is a synthetic chain molecule. A UNA oligomer of this invention is not a nucleic acid, nor an oligonucleotide.
  • a UNA monomer can be UNA-A (designated A), UNA-U (designated U), UNA-C (designated C), and UNA-G (designated G).
  • Designations that may be used herein include mA, mG, mC, and mU, which refer to the 2'-0-Methyl modified ribonucleotides.
  • Designations that may be used herein include lower case c and u, which refer to the 2'-0-methyl modified ribonucleotides.
  • Designations that may be used herein include dT, which refers to a 2'- deoxy T nucleotide.
  • N represents any natural nucleotide monomer, or a modified nucleotide monomer.
  • the symbol Q represents a non-natural, modified, or chemically-modified nucleotide monomer.
  • the monomer can have any base attached.
  • the Q monomer can have any base attached that would be complementary to the monomer in the corresponding paired position in the other strand.
  • nucleic acid monomers include non-natural, modified, and chemically-modified nucleotides, including any such nucleotides known in the art.
  • non-natural, modified, and chemically-modified nucleotide monomers include 2'-0-methyl ribonucleotides, 2'-0-methyl purine nucleotides, 2'- deoxy-2'-fluoro ribonucleotides, 2'-deoxy-2'-fluoro pyrimidine nucleotides, 2'-deoxy ribonucleotides, 2'-deoxy purine nucleotides, universal base nucleotides, 5-C-methyl- nucleotides, and inverted deoxyabasic monomer residues.
  • non-natural, modified, and chemically-modified nucleotide monomers include 3 '-end stabilized nucleotides, 3 '-glyceryl nucleotides, 3 '-inverted abasic nucleotides, and 3 '-inverted thymidine, and L-thymidine.
  • non-natural, modified, and chemically-modified nucleotide monomers include locked nucleic acid nucleotides, 2'-0,4'-C-methylene-(D- ribofuranosyl) nucleotides, 2'-methoxyethoxy (MOE) nucleotides, 2'-methyl-thio-ethyl, 2'-deoxy-2'-fluoro nucleotides, and 2'-0-methyl nucleotides.
  • locked nucleic acid nucleotides 2'-0,4'-C-methylene-(D- ribofuranosyl) nucleotides
  • MOE methoxyethoxy
  • Examples of non-natural, modified, and chemically-modified nucleotide monomers include 2'-amino nucleotides, 2'-0-amino nucleotides, 2'-C-allyl nucleotides, and 2'-0-allyl nucleotides.
  • non-natural, modified, and chemically-modified nucleotide monomers include N 6 -methyladenosine nucleotides.
  • nucleotide monomers examples include nucleotide monomers with modified bases 5-(3-amino)propyluridine, 5-(2-mercapto)ethyluridine, 5-bromouridine; 8-bromoguanosine, or 7-deazaadenosine.
  • non-natural, modified, and chemically-modified nucleotide monomers include 2'-0-aminopropyl substituted nucleotides.
  • non-natural, modified, and chemically-modified nucleotide monomers include 2'-0-guanidinopropyl substituted nucleotides.
  • Examples of non-natural, modified, and chemically-modified nucleotide monomers include Pseudouridines.
  • Examples of non-natural, modified, and chemically-modified nucleotide monomers include replacing the 2'-OH group of a nucleotide with a 2'-R, a 2'-OR, a 2'- halogen, a 2'-SR, or a 2'-amino, or a 2'-azido, where R can be H, alkyl, fluorine- substituted alkyl, alkenyl, or alkynyl.
  • Examples of non-natural, modified, and chemically-modified nucleotide monomers include replacing the 2'-OH group of a nucleotide with a 2'-R or 2'-OR, where R can be CN, CF 3 , alkylamino, or aralkyl.
  • non-natural, modified, and chemically-modified nucleotide monomers include nucleotides with a modified sugar such as an F-HNA, an HNA, a CeNA, a bicyclic sugar, or an LNA.
  • a modified sugar such as an F-HNA, an HNA, a CeNA, a bicyclic sugar, or an LNA.
  • Examples of non-natural, modified, and chemically-modified nucleotide monomers include 2'-oxa-3'-aza-4'a-carbanucleoside monomers, 3-hydroxymethyl-5- (lH-l,2,3-triazol)-isoxazolidine monomers, and 5'-triazolyl-2'-oxa-3'-aza-4'a- carbanucleoside monomers.
  • aspects of this invention can provide structures and compositions for UNA-containing oligomeric compounds.
  • the oligomeric agents may incorporate one or more UNA monomers.
  • Oligomeric molecules of this invention can be used as active agents in formulations for gene regulating or gene silencing therapeutics.
  • this invention provides oligomeric compounds having a structure that incorporates novel combinations of UNA monomers with certain natural nucleotides, non-natural nucleotides, modified nucleotides, or chemically- modified nucleotides.
  • the oligomeric compounds can be pharmacologically active molecules.
  • UNA oligomers of this invention can be used as active pharmaceutical ingredients for regulating gene expression, and in RNA interference methods, as well as antisense, RNA blocking, and micro-RNA strategies.
  • a UNA oligomer of this invention can be a short chain molecule.
  • a UNA oligomer can be a duplex pair.
  • a UNA oligomer can have a first strand of the duplex and a second strand of the duplex, which is complementary to the first strand with respect to the nucleobases, although up to three mismatches can occur.
  • a UNA oligomer duplex can have overhangs.
  • the target of a UNA oligomer can be a target nucleic acid.
  • the target can be any TTR mRNA of a subject.
  • a UNA oligomer can be active for gene silencing in RNA interference.
  • a UNA oligomer may comprise two strands that together provide a duplex.
  • the duplex may be composed of a first strand, which may also be referred to as a passenger strand or sense strand, and a second strand, which may also be referred to as a guide strand or antisense strand.
  • a UNA oligomer of this invention can have any number of phosphorothioate intermonomer linkages in any position in any strand, or in both strands of a duplex structure.
  • a UNA oligomer of this invention can have a phosphorothioate intermonomer linkage between the last one or two monomers at either end of any strand.
  • any one or more of the intermonomer linkages of a UNA oligomer can be a phosphodiester, a phosphorothioate including dithioates, a chiral phosphorothioate, and other chemically modified forms.
  • Examples of UNA oligomers of this invention include duplex pairs, which are in general complementary.
  • SEQ ID NO: l can represent a first strand of a duplex and SEQ ID NO:2 can represent a second strand of the duplex, which is complementary to the first strand.
  • N in the first strand can represent any nucleotide that is complementary to the monomer in the corresponding position in the second strand.
  • Example UNA oligomers of this disclosure are shown with 2-monomer length overhangs, although overhangs of from 1 to 8 monomers, or longer, can be used.
  • the symbol "X" in a strand or oligomer represents a UNA monomer.
  • the monomer can have any base attached.
  • the UNA monomer can have any base attached that would be complementary to the monomer in the corresponding paired position in the other strand.
  • the terminal position has a 1-end, according to the positional numbering shown above, instead of a 5'- end as for a nucleotide, or the terminal position has a 3 -end, according to the positional numbering shown above, instead of a 3 '-end as for a nucleotide.
  • the UNA oligomer terminates in a UNA monomer, the terminal position has a 1-end, according to the positional numbering shown above, instead of a 5'- end as for a nucleotide, or the terminal position has a 3 -end, according to the positional numbering shown above, instead of a 3 '-end as for a nucleotide.
  • the UNA oligomer terminates in a UNA monomer
  • complementarity of strands can involve mismatches.
  • complementarity of strands can include one to three, or more, mismatches.
  • a UNA oligomer of this invention can have one or more UNA monomers at the 1-end of the first strand, and one or more UNA monomers at the 3 -end of the first strand.
  • a UNA oligomer of this invention can have one or more UNA monomers at the 3 -end of the second strand.
  • a duplex UNA oligomer of this invention can have one or more UNA monomers at the 1 -end of the first strand, one or more UNA monomers at the 3 -end of the first strand, and one or more UNA monomers at the 3 -end of the second strand.
  • a UNA oligomer of this invention may have a first strand and a second strand, each of the strands independently being 19-23 monomers in length.
  • a UNA oligomer of this invention may have a first strand that is 19-23 monomers in length.
  • a UNA oligomer of this invention may have a duplex region that is 19-21 monomers in length.
  • a UNA oligomer of this invention may have a second strand that is 19-23 monomers in length.
  • a UNA oligomer of this invention may have a first strand that is 19 monomers in length, and a second strand that is 21 monomers in length.
  • a UNA oligomer of this invention may have a first strand that is 20 monomers in length, and a second strand that is 21 monomers in length.
  • a UNA oligomer of this invention may have a first strand that is 21 monomers in length, and a second strand that is 21 monomers in length.
  • a UNA oligomer of this invention may have a first strand that is 22 monomers in length, and a second strand that is 21 monomers in length.
  • a UNA oligomer of this invention for inhibiting gene expression can have a first strand and a second strand, each of the strands being 19-29 monomers in length.
  • the monomers can be UNA monomers and nucleic acid nucleoside monomers.
  • the oligomer can have a duplex structure of from 14 to 29 monomers in length.
  • the UNA oligomer can be targeted to a target gene and can exhibit reduced off-target effects as compared to a conventional siRNA.
  • a UNA oligomer of this invention can have a first strand and a second strand, each of the strands being 19-23 monomers in length.
  • the UNA oligomer may have a blunt end, or may have one or more overhangs.
  • the first and second strands may be connected with a connecting oligomer in between the strands, and form a duplex region with a connecting loop at one end.
  • an overhang can be one or two monomers in length.
  • Examples of an overhang can contain one or more UNA monomers, natural nucleotides, non-natural nucleotides, modified nucleotides, or chemically- modified nucleotides, and combinations thereof.
  • Examples of an overhang can contain one or more deoxythymidine nucleotides, 2'-0-methyl nucleotides, inverted abasic monomers, inverted thymidine monomers, L-thymidine monomers, or glyceryl nucleotides.
  • a UNA oligomer can mediate cleavage of a target nucleic acid in a cell.
  • the second strand of the UNA oligomer at least a portion of which can be complementary to the target nucleic acid, can act as a guide strand that can hybridize to the target nucleic acid.
  • the second strand can be incorporated into an RNA Induced Silencing Complex (RISC).
  • RISC RNA Induced Silencing Complex
  • a UNA oligomer may have a strand that is a DICER substrate.
  • a UNA oligomer of this disclosure may comprise naturally-occurring nucleic acid nucleotides, and modifications thereof that are compatible with gene silencing activity.
  • a UNA oligomer is a double stranded construct molecule that is able to inhibit gene expression.
  • strand refers to a single, contiguous chain of monomers, the chain having any number of internal monomers and two end monomers, where each end monomer is attached to one internal monomer on one side, and is not attached to a monomer on the other side, so that it ends the chain.
  • the monomers of a UNA oligomer may be attached via phosphodiester linkages, phosphorothioate linkages, gapped linkages, and other variations.
  • a UNA oligomer can include mismatches in complementarity between the first and second strands.
  • a UNA oligomer may have 1, or 2, or 3 mismatches. The mismatches may occur at any position in the duplex region.
  • the target of a UNA oligomer can be a target nucleic acid of a target gene.
  • a UNA oligomer may have one or two overhangs outside the duplex region.
  • the overhangs can be an unpaired portion at the end of the first strand or second strand.
  • the lengths of the overhang portions of the first and second strands can be the same or different.
  • a UNA oligomer may have at least one blunt end.
  • a blunt end does not have an overhang portion, and the duplex region at a blunt end terminates at the same position for both the first and second strands.
  • a UNA oligomer can be RISC length, which means that it has a duplex length of less than 25 base pairs.
  • a UNA oligomer can be a single strand that folds upon itself and hybridizes to itself to form a double stranded region having a connecting loop at the end of the double stranded region.
  • Ref Pos refers to reference position, which is the numerical position of a reference nucleotide in a TTR mRNA.
  • a UNA oligomer would be composed of pairs SEQ ID NOs:23 and 63, 24 and 64, etc.
  • lower case designates 2'-deoxyribo-N; upper case designates Ribo-N; underlined-lower case designates 2'-deoxy-N; mA, mG, mC, and mU designate 2'-0-Methyl RNA; and A ⁇ G and C designate UNA monomers.
  • a UNA oligomer may have a duplex region, and have a UNA monomer in the second strand within the duplex region, where the UNA monomer in the second strand is present in any of positions 1 through 19, counting from the 5' end of the second strand.
  • a UNA oligomer may have a duplex region, and have a UNA monomer in the second strand within the duplex region, where the UNA monomer in the second strand is present in any of positions 6, 7, 14, 15 and 16, counting from the 5' end of the second strand.
  • a UNA oligomer may comprise an overhang portion of two monomers in length, or longer, at the 3' end of the first strand, wherein the overhang monomer immediately flanking the duplex portion is a UNA monomer.
  • a UNA oligomer may comprise an overhang portion of two monomers in length, or longer, at the 3' end of the second strand, wherein the overhang monomer immediately flanking the duplex portion is a UNA monomer.
  • Methods of this invention include the treatment and prevention of TTR- related amyloidosis in human and mammalian subjects.
  • a subject in need of treatment or prevention can be administered an effective amount of a UNA oligomer.
  • Administration can be performed for 1, 2, or up to 7 days, or 1, 2, 3, or up to 4 weeks, or longer.
  • the subject may have TTR-related amyloidosis, also known as ATTR.
  • a subject can have a V30M gene.
  • the methods of this invention can selectively reduce V30M TTR in the subject.
  • a method of this invention can selectively reduce V30M TTR in the subject by at least 10%, as compared to control. In certain embodiments, V30M TTR in the subject can be reduced by at least 20%, or 30%, or 50%, as compared to control.
  • An effective amount of a UNA oligomer of this invention can be a dose ranging from 0.001 mg/kg to 50.0 mg/kg, or from 0.001 mg/kg to 10.0 mg/kg.
  • TTR mRNA expression can be reduced in a subject for at least 5 days. In certain embodiments, TTR mRNA expression can be reduced in a subject for at least 10 days, or 15 days.
  • peripheral neuropathy or autonomic neuropathy in the subject can be reduced.
  • peripheral neuropathy or autonomic neuropathy in the subject can be reduced.
  • a subject may undergo reduced lower extremity weakness, reduced pain, or improved sensation.
  • Methods of this invention can reduce occurrence of vitreous opacities in the subject.
  • the administration of a UNA oligomer may not result in an inflammatory response.
  • this invention includes methods for inhibiting expression of a TTR gene in a cell, by treating the cell with a UNA oligomer.
  • this invention includes methods for inhibiting expression of a TTR gene in a mammal, by administering to the mammal a composition containing a UNA oligomer.
  • this invention provides compositions containing a UNA oligomer and a pharmaceutically acceptable carrier.
  • this invention includes nanoparticle compositions that can encapsulate and deliver a UNA oligomer to cells with surprisingly advantageous potency.
  • the nanoparticles can be formed with lipid molecules, for example, any one or more of the compounds ATX-001 to ATX-032 disclosed in WO/2015/074085.
  • lipid nanoparticles of this invention can be formed with compound ATX- 002, as disclosed in WO/2015/074085, and the nanoparticles can encapsulate the UNA oligomer.
  • a composition can be capable of local or systemic administration.
  • a pharmaceutical composition can be capable of any modality of administration.
  • the administration can be intravenous, subcutaneous, pulmonary, intramuscular, intraperitoneal, dermal, oral, or nasal administration.
  • Embodiments of this invention include pharmaceutical compositions containing a UNA oligomer in a lipid formulation.
  • a composition may comprise one or more lipids selected from cationic lipids, anionic lipids, sterols, pegylated lipids, and any
  • a composition can be substantially free of liposomes.
  • a composition can include liposomes.
  • a composition can contain a UNA oligomer within a viral or bacterial vector.
  • a pharmaceutical composition of this disclosure may include carriers, diluents or excipients as are known in the art. Examples of pharmaceutical compositions are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro ed. 1985).
  • excipients for a pharmaceutical composition include antioxidants, suspending agents, dispersing agents, preservatives, buffering agents, tonicity agents, and surfactants.
  • Example 1 shows the single nucleotide polymorph (SNP) that exists at position 284 in the V30M mutation of the human TTR mRNA, as compared to the wild type (WT) TTR mRNA.
  • SNP single nucleotide polymorph
  • Conventional siRNAs that are complementary to the WT mRNA were tiled around position 284.
  • Fig. 2 shows that the conventional siRNAs complementary to the WT mRNA have limited activity in silencing the WT TTR gene, as measured by TTR knockdown in HepG2 cells. Positions 5, 9, 14 and 15 appear to be more accessible to silencing than other positions.
  • Fig. 3 shows conventional siRNAs that were complementary to the V30M mRNA were tiled around position 284.
  • V30M-P5 Four conventional siRNA variations, namely V30M-P5, V30M-P9, V30M-P14, and V30M- P15, were prepared. Also, as shown in Fig. 3, two gene reporter variants, V30V and V30M, each bearing nucleotide sequence 264 to 304 of human TTR, V30V being without the point mutation at position 284, and V30M containing the point mutation at position 284, were prepared and used in the PSICHECK reporter system in the 3'-UTR region of Luciferase gene.
  • Example 2 shows the activity of the four conventional siRNA variations V30M-P5, V30M-P9, V30M-P14, and V30M-P15 as measured in the
  • FIG. 5 shows IC50 analysis for the four conventional siRNA variations V30M-P5, V30M-P9, V30M-P14, and V30M-P15 measured in the PSICFIECK reporter assay against V30V and V30M gene reporter variants.
  • the conventional V30M-P15 variant was 5.6 times more effective against V30M than V30V. Thus, the selectivity of the conventional siRNAs against V30M over V30V was no more than 5.6.
  • Example 3 shows the structure of UNA oligomers that were effective in silencing V30M TTR, as measured in the PSICHECK reporter assay.
  • a first UNA monomer was located at the 5' end of the first strand, also called the passenger strand.
  • the second strand also called the guide strand, formed a duplex region of 19 monomers length with the first strand.
  • Each UNA oligomer had a duplex region of 19 monomers, and a two-monomer overhang at each end.
  • a second UNA monomer was located at the 3' end of the first strand, in the 20 th position, which is in an overhang portion.
  • a third UNA monomer was located at the 3' end of the second strand, in the 20 th position, which is in an overhang portion.
  • P15U6, P15U7, P15U14, P15U15, and P15U16 a fourth UNA monomer was located in the second strand at positions 6, 7, 14, 15 and 16, respectively, counting from the 5' end of the second strand.
  • Example 4 shows the activity of UNA oligomers P15U6, P15U7, P15U15, and P15U16, measured in the PSICHECK reporter assay against V30V and V30M gene reporter variants, as compared to the conventional siRNA V30M-P15.
  • the UNA oligomers were more effective against V30M than V30V.
  • the activity of the UNA oligomer P15U6 was substantially and advantageously superior to the activity of the conventional siRNA V30M-P15, where each is targeted to V30M.
  • Fig. 7 shows the surprising and unexpected result that the selectivity of the UNA oligomers, P15U6, P15U7, P15U15, and P15U16, against V30M over V30V was substantially greater than for the conventional siRNA V30M-P15.
  • the selectivity of UNA oligomer PI 5U6 against V30M over V30V was 24, meaning that the IC50 of UNA oligomer P15U6 against V30M (37.6 pM) was 24 times lower than the IC50 of UNA oligomer P15U6 against V30V (919.9 pM).
  • This selectivity was advantageously 4-fold superior to the selectivity of 5.6 shown by the conventional siRNA.
  • Example 5 shows the structure of UNA oligomers that were effective in silencing V30M TTR, as measured in the PSICHECK reporter assay.
  • a first UNA monomer was located at the 5' end of the first strand, also called the passenger strand.
  • the second strand also called the guide strand, formed a duplex region of 19 monomers length with the first strand.
  • Each UNA oligomer had a duplex region of 19 monomers, and a two-monomer overhang at each end.
  • a second UNA monomer was located at the 3' end of the first strand, in the 20 th position, which is in an overhang portion.
  • a third UNA monomer was located at the 3' end of the second strand, in the 20 position, which is in an overhang portion.
  • P16U6, P16U7, P16U15, P16U16, and P16U17 a fourth UNA monomer was located in the second strand at positions 6, 7, 15, 16 and 17, respectively, counting from the 5' end of the second strand.
  • Example 6 shows the activity of UNA oligomers P16U6, P16U7, P16U15, and P16U16, measured in the PSICHECK reporter assay against V30V and V30M gene reporter variants, as compared to the conventional siRNA V30M-P16.
  • the UNA oligomers were more effective against V30M than V30V.
  • each of the UNA oligomers P16U6, P16U7, P16U15, and P16U16 was substantially and advantageously superior to the activity of the conventional siRNA V30M-P16, where each is targeted to V30M.
  • Fig. 9 shows the surprising and unexpected result that the selectivity of the UNA oligomers, P16U6, P16U7, P16U15, and P16U16, against V30M over V30V was substantially greater than for the conventional siRNA V30M-P16.
  • the selectivity of UNA oligomer P16U6 against V30M over V30V was 23, meaning that the IC50 of UNA oligomer PI 6U6 against V30M (92.4 pM) was 23 times lower than the IC50 of UNA oligomer P16U6 against V30V (2119 pM).
  • This selectivity was advantageously 4-fold superior to the selectivity of 5.6 shown by the conventional siRNA.
  • Fig. 10 shows the selectivity of UNA oligomer P15U6 against V30M over V30V.
  • the IC50 of UNA oligomer P15U6 against V30M 37.6 pM
  • This selectivity was advantageously 4-fold superior to the selectivity of 5.6 shown by the conventional siRNA.
  • Fig. 10 (right) shows the surprising and unexpected result that the IC50 of UNA oligomer P16U6 against V30M (92.4 pM) was 23 times lower than the IC50 of UNA oligomer P16U6 against V30V (2119 pM).
  • This selectivity was advantageously 4-fold superior to the selectivity of 5.6 shown by the conventional siRNA.
  • Example 7 UNA oligomers reduce V30M TTR deposits in vivo
  • Transgenic mice for human TTR V30M overexpression are used at 6 months age.
  • TTR wild-type and TTR knockout mice are used as controls. Animals are housed in controlled environment, and euthanized with ketamine and medetomidine.
  • Liver and colon mRNA are isolated using phenol extraction (Invitrogen). Sciatic nerve from V30M mice is dissected from other tissue, and mRNA is extracted with a RNeasy Mini column (Qiagen). cDNA is synthesized with a Superscript double- stranded cDNA Kit (Invitrogen). Extracted RNA is validated with Experion RNA StdSens Analysis Kit (Bio-Rad). qPCR is performed with primers and iQ Syber Green Super Mix (Bio-Rad). Double immunofluorescence analysis is performed with sciatic nerve, dorsal root ganglia, and colon from V30M animals that is removed and treated as above. Comparisons are performed with Student T-test or One-way ANOVA. Data are expressed as mean values ⁇ standard error (SEM). ⁇ -values less than 0.05 are considered significant.
  • Example 8 UNA oligomers reduce TTR protein in vivo primate.
  • Transthyretin protein was evaluated as a result of treatment with UNA oligomers targeting Transthyretin TTR mRNA, based on SEQ ID NOs:46/86, 59/99, and 60/100, delivered in a liposomal formulation to male Cynomolgus monkeys. Measurements were made following a single intravenous (IV) dose administration. TTR protein concentration in plasma samples was assessed by LC- MS/MS at various time points.
  • Animals in Groups 1 through 8 were administered the test articles on Day 1 at 0.3 mg/kg, dose volume 6.25 mL/kg, concentration 0.048 mg/mL. Doses were administered as a 15-minute intravenous (IV) infusion, ⁇ 1 minute, using a pump and disposable syringe.
  • IV intravenous
  • Fig. 11 shows the unexpectedly advantageous result of over 91% knockdown of TTR in the primates at 20 days, and more than 70% knockdown with a continuous duration of more than 20 days.
  • the Monkey TTR SRM assay was used, which is a high throughput absolute protein quantitation assay using Selected Reaction Monitoring (SRM) technology platform.
  • SRM Selected Reaction Monitoring
  • the panel can multiplex up to 200 proteins.
  • the assay was performed on a targeted absolute protein quantitation platform, where the key technology was SRM, sometimes also referred to as Multiple Reaction Monitoring (MRM).
  • MRM Multiple Reaction Monitoring
  • SRM/MRM is a tandem selection process where selecting peptides by precursor m/z is followed by selecting fragmentations (transitions) of the targeted peptide.
  • QQQQ triple-quadrupole
  • SRM/MRM is a tandem selection process where selecting peptides by precursor m/z is followed by selecting fragmentations (transitions) of the targeted peptide.
  • SRM/MRM is a highly selective, sensitive technology to quantify designated protein with precision.

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Abstract

La présente invention concerne des composés et des compositions utiles dans des procédés permettant une thérapie médicale, en général, pour inhiber l'expression d'un gène de transthyrétine (TTR) chez un sujet. Les composés ont un premier brin et un second brin, chaque brin ayant une longueur de 19 à 29 monomères, les monomères comprenant des monomères UNA et des monomères d'acide nucléique et les composés sont ciblés pour une séquence d'un gène de transthyrétine.
PCT/US2016/043927 2015-07-23 2016-07-25 Compositions permettant de traiter l'amylose WO2017015671A1 (fr)

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